Figure 3. PI3Kγ is promotes inflammation in response to SARS-CoV-2 infection.

(A) Graph of PI3KCG gene transcripts determined by RNA sequencing of FFPE lung tissue from normal (n=5) and COVID-19 patients (n=14). (B) Images of CD68 and PI3Kγ expression in lung tissue from COVID-19 patients. Scale bar indicates 25 μm. Arrowheads indicate CD68⁺ and PI3Kγ⁺ cells in serial 5 μm sections. (C) PI3Kγ (red), CD68 (green), and DAPI (blue) confocal (upper panels) and stochastic optical reconstruction microscopy (STORM, lower panels) images of macrophages in COVID-19 infected lungs. Scale bars indicate 25 μm or 75 μm (upper) and 2 μm or 10 μm (lower). Insets indicate fields magnified in subsequent panels. Arrowheads identify CD68⁺PI3Kγ⁺ stained cells. (D) Immunostaining for MPO⁺ granulocytes, IBA1⁺ macrophages and RNAscope detection of PI3Kγ mRNA in lung tissue from uninfected (PBS) and SARS-CoV-2 infected Syrian golden hamsters. Scale bar indicates 10μm. Arrowheads identify stained cells. (E) Graphs of mean ± SEM IBA1⁺ macrophages, MPO+ granulocytes and PI3Kγ mRNA⁺ cells in lung tissue from uninfected (PBS, n=2–5) and SARS-CoV-2 infected (COVID, n=3–4) hamsters. (F) Schematic of hamster infection with SARS-CoV-2 and treatment with vehicle (n=4) or IPI-549 (n=4). (G and H) Images of IBA1+ macrophages (G) and MPO+ granulocytes (H) in lungs from (F). (I) Graphs of mean ± SEM MPO⁺ neutrophils and IBA1⁺ macrophages in hamster lungs over time. Arrows indicate days of treatment. (J) Schematic of K18-ACE2 transgenic mice infected with SARS-CoV-2 and treated with vehicle or IPI-549. (K) Graph of median weight loss over time for mice infected with SARS-CoV-2 and treated with vehicle (n=10) or IPI-549 (n=10). (L) Graph of mean ± SEM viral load expressed as focus-forming units (ffu)/gm lung tissue in mice (n=10 per group) from (J). (M) Images of H&E, MPO⁺ and F4/80+ staining in endpoint tissues from (K). Scale bar indicates 20 μm. Arrowheads indicate F4/80⁺ macrophages and MPO⁺ granulocytes. (N and O) Graphs of mean ± SEM F4/80⁺ macrophages/mm² (N) and MPO+ granulocytes/mm² (O) in lung tissue from mice infected with SARS-CoV-2 and treated with vehicle (n=5) or IPI-549 (n=5). Statistical analysis was performed by limma-vroom (A), unpaired T tests (K, L, N, O), or one-way ANOVA with Tukey’s post hoc multiple comparisons test (E, I).